10 Mbp of oligos / $300 chip
8K Atactic/Xeotron/Invitrogen
Photo-Generated Acid
12K Combimatrix Electrolytic
44K Agilent Ink-jet standard reagents
380K Nimblegen/GA Photolabile 5'protection
~1000X lower oligo costs
Amplify pools of 50mers using flanking universal PCR primers & 3 paths to 10X error correction
Tian et al. Nature. 432:1050 Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS
ss-Oligonucleotide Repair
Obtain 25% recombination efficiency in E. coli strains lacking mismatch repair genes (mutH, mutL, mutS, uvrD, dam)
Ellis et al. PNAS 2001Constantino & Court. PNAS 2003
DNA Replication Fork
Improved Recombination Frequency:10-4 0.25 (> 3 log increase!)
Multiplex Automated Genome Engineering (MAGE)
Wash with water &
DNA pool (50)
Concentrate, electroporate
Resuspend, bubble, select
O-ring membrane
Concentrate
Wang, Isaacs, Terry
Multiplex Automated Genome Engineering (MAGE)
syringe pump
electrically actuated valves
electroporation cuvette w/ membrane filter
OD sensor
data acquisition systemcomputer communication /
Wang, Isaacs, Terry
rE.coli: new in vivo genetic codes
TTT
F
30362 TCT
S
11495 TAT
Y
21999 TGT
C
7048
TTC 22516 TCC 11720 TAC 16601 TGC 8816
TTA
L
18932 TCA 9783 TAASTOP
STOP
2703 TGA STOP 1256
TTG 18602 TCG 12166 TAG 326 TGG W 20683
CTT
L
15002 CCT
P
9559 CAT
H
17613 CGT
R
28382
CTC 15077 CCC 7485 CAC 13227 CGC 29898
CTA 5314 CCA 11471 CAA
Q
20888 CGA 4859
CTG 71553 CCG 31515 CAG 39188 CGG 7399
ATT
I
41309 ACT
T
12198 AAT
N
24159 AGT
S
11970
ATC 34178 ACC 31796 AAC 29385 AGC 21862
ATA 5967 ACA 9670 AAA
K
45687 AGA
R
2896
ATG M 37915 ACG 19624 AAG 14029 AGG 1692
GTT
V
24858 GCT
A
20762 GAT
D
43719 GGT
G
33622
GTC 20753 GCC 34695 GAC 25918 GGC 40285
GTA 14822 GCA 27418 GAA
E
53641 GGA 10893
GTG 35918 GCG 45741 GAG 24254 GGG 15090
Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGRe.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability
IsaacsWang
Church
Forster
CarrJacobson
1
2
3
4